Efficient refolding of proteins in vitro is an important problem in protein structural analysis and biotechnological manufacturing of pharmaceutical products. Because of their inherent ability to rapidly overexpress proteins to high yields, bacterial systems are the organisms of choice for protein mass production. Unfortunately, overexpression of foreign and, especially, mutant proteins often leads to the development of large intracellular aggregates or inclusion bodies (Rudolph et al., In vitro folding of inclusion body proteins, FASEB J. 10 49-56 (1996); Guise et al., Protein folding in vivo and renaturation of recombinant proteins from inclusion bodies, Mol. Biotechnol. 6 53-64 (1996), the disclosures of which are incorporated herein by reference). In some cases, the proper intracellular folding of the overexpressed proteins can be enhanced by lowering the cell growth temperature, co-expressing molecular chaperones, or introducing low molecular weight additives (Kujau et al., Expression and secretion of functional miniantibodies McPC603scFvDhlx in cell wall-less L-form strains of Proteus mirabilis and E. coli, Appl. Microbiol. Biotechnol. 49 51-58 (1998); Tate et al., Molecular Chaperones Stimulate the Functional Expression of the Cocaine-sensitive Serotonin Transporter, J. Biol-Chem. 274 17551-17558 (1999); Minning et al., Functional expression of Rhizopus oryzae lipase in Pichia pastoris: high-level production and some properties, J. Biotechnol. 66 147-156 (1998), the disclosures of which are incorporated herein by reference). More often, however, investigators are forced to rely on in vitro folding methods to denature (also known as “deactivate”) and then refold (also known as “reactivate”) aggregated proteins. A number of in vitro approaches have been developed to minimize protein aggregation and enhance proper refolding. Among those are: (1) the addition of osmolytes and denaturants to refolding buffer (Tate et al., Molecular Chaperones Stimulate the Functional Expression of the Cocaine-sensitive Serotonin Transporter, J. Biol-Chem. 274 17551-17558 (1999); Plaza-del-Pino et al., An osmolyte effect on the heat capacity change for protein folding, Biochemistry 34 8621-8630 (1995); Frye et al., The kinetic basis for the stabilization of staphylococcal nuclease by xylose, Protein. Sci. 6 789-793 (1997), the disclosures of which are incorporated herein by reference); (2) the use of the combinations of different molecular chaperones (Thomas et al., Molecular chaperones, folding catalysts, and the recovery of active recombinant proteins from E. coli. To fold or to refold, Appl. Biochem. Biotechnol. 66 197-238 (1997); Buchberger, A., Schroder, H., Hesterkamp, T., Schonfeld, H. J., and Bukau, B. (1996) J. Mol. Biol. 261, 328-233; Veinger et al., The Small Heat-shock Protein IbpB from Escherichia coli Stabilizes Stress-denatured Proteins for Subsequent Refolding by a Multichaperone Network, J. Biol. Chem. 273, 11032-11037 (1998), the disclosures of which are incorporated herein by reference); (3) immobilization of folding proteins to matrices and matrix-bound chaperonins (Stempfer, G., Holl-Neugebauer, B., and Rudolph, R. (1996) Nat. Biotechnol. 14, 329-334; Proc. Natl. Acad. Sci. USA 94, 3576-3578; Preston, N. S., Baker, D. J., Bottomley, S. P., and Gore, M. G. (1999) Biochim. Biophys. Acta 1426, 99-109, the disclosures of which are incorporated herein by reference); and (4) utilization of folding catalysts such as protein disulfide isomerase and peptidyl-prolyl cis-trans isomerase (Altamirano, M. M., Garcia, C., Possani, L. D., and Fersht, A. R. (1999) Nat. Biotechnol. 17, 187-191, the disclosure of which is incorporated herein by reference). While the latter investigators (Altamirano et al., 1999) used a truncated monomer of the chaperonin, prominent researchers in the field have since demonstrated that the best functional construction of the chaperonin is its native oligomeric form (Wang, J D, Michelitsch M D, and Weissman J S (1998) “GroEL-GroES-mediated protein folding requires and intact central cavity” Proc. Natl. Acad. Sci. USA 95, 12163-12168; Weber F, Keppel F, Georgopoulos C, Hayer-Hartl M K, Hartl F U. The oligomeric structure of GroEL/GroES is required for biologically significant chaperonin function in protein folding, Nat Struct Biol. 5(11):977-85). Because of the diversity of the protein folding mechanisms, there has been no universal procedure for protein folding and folding conditions have to be optimized for each specific protein of interest. Therefore, there is always a need for new and more versatile folding techniques. This invention involves a novel protein folding procedure that utilizes a novel stabilizing osmolyte composition useful for stabilizing protein intermediates. Chaperonins, particularly the functional and tight binding oligomeric chaperonins (e.g., GroE), can be added in order to facilitate complete folding of the protein to its native functional form.
Because of its ability to bind many different protein folding intermediates, it was thought that the bacterial GroE chaperonin system could provide a general method to refold misfolded proteins. Chaperonin GroEL is a tetradecamer of identical 57 kDa subunits that possesses two large hydrophobic sites capable of binding to transient hydrophobic protein folding intermediates. The hydrophobic binding site undergoes the multiple cycles of exposure and burial driven by the ATP binding and hydrolysis and the co-chaperonin GroES binding and dissociation. Accordingly, the protein folding intermediates can undergo multiple rounds of binding to and release from the GroEL until they achieve the correctly folded state (for review, see Fenton, W. A. and Horwich, A. L. (1997) Protein Sci. 6, 743-760, the disclosure of which is incorporated herein by reference). Besides simple prevention of non-productive aggregation, chaperonins may also influence the conformation of the folding intermediates, actively diverting them to a productive folding pathway (Fedorov, A. N. and Baldwin, T. O. (1997) J. Mol. Biol. 268, 712-723; Shtilerman, M., Lorimer, G., and Englander, S. W. (1999) Science 284, 822-825, the disclosures of which are incorporated herein by reference). However, despite the general nature of chaperonin-protein interactions, there are many proteins that, for reasons that are currently unknown, cannot fold correctly from the bacterial chaperonin system.
The addition of osmolytes often results in an observed increase in stability of the native structure for some proteins. The stabilization effect is observed with various osmolytes and small electrolytes such as sucrose, glycerol, trimethylamine N-oxide (TMAO), potassium glutamate, arginine and betaine (Wang, A. and Bolen, D. W. (1997) Biochemistry 36, 9101-9108; De-Sanctis, G., Maranesi, A., Ferri, T., Poscia, A., Ascoli, F., and Santucci, R. (1996) J. Protein. Chem. 15, 599-606; Chen, B. L. and Arakawa, T. (1996) J. Pharm. Sci. 85, 419-426; Zhi, W., Landry, S. J., Gierasch, L. M., and Srere, P. A. (1992) Protein Science 1, 552-529, the disclosures of which are incorporated herein by reference). This effect is based on the exclusion of osmolytes from hydration shells and crevices on protein surface (Timasheff, S. N. (1992) Biochemistry 31 9857-9864, the disclosure of which is incorporated herein by reference) or decreased solvation (Parsegian, V. A., Rand, R. P., and Rau D. (1995) Methods. Enzymol. 259 43-94, the disclosure of which is incorporated herein by reference). In a series of quantitative studies, Wang and Bolen have shown that the osmolyte-induced increase in protein stability is due to a preferential burial of the polypeptide backbone rather than the amino acid side chains (Wang, A. and Bolen, D. W. (1997) Biochemistry 36 9101-9108; Bolen et al., The Osmophobic Effect: Natural Selection of a Thermodynamic Force in Protein Folding, J. Mol. Biol. Vol. 310 955-963 (2001)). Because native protein conformations are stabilized, proper folding reactions are also enhanced in the presence of osmolytes (Frye, K. J. and Royer, C. A. (1997) Protein. Sci. 6 789-793; Kumar, T. K., Samuel, D., Jayaraman, G., Srimathi, T., and Yu, C. (1998) Biochem. Mol. Biol. Int. 46 509-517; Baskakov, I. and Bolen, D. W. (1998) J. Biol. Chem. 273 4831-4834, the disclosures of which are incorporated herein by reference). Osmolytes usually affect protein stability and folding at physiological concentration range of 1-4 M (Yancey, P. H., Clark, M. E., Hand, S. C., Bowlus, R. D., and Somero, G. N. (1982) Science 217 1214-1222, the disclosure of which is incorporated herein by reference). However, it is apparent that the degree of stabilization depends on both the nature of the osmolyte and the protein substrate (Sola-Penna, M., Ferreira-Pereira, A., Lemos, A. P., and Meyer-Fernandes, J. R. (1997) Eur. J. Biochem. 248 24-29, the disclosure of which is incorporated herein by reference) and, in some instances, the initial aggregation reaction can actually accelerate in the presence of some strong folding osmolytes such as TMAO (Voziyan, P. A. and Fisher M. T. (2000) Protein Science, Vol 9 2405-2412).
Fisher et al., U.S. Pat. No. 6,887,682, which is incorporated by reference, found that folding of a denatured polypeptide could be improved by first forming a chaperonin-polypeptide complex, and then adding an osmolyte to promote folding. Despite the advances set forth in the Fisher '682 patent, improved compositions of matter and methods for stabilizing and folding denatured proteins are needed.